Method for quantifying a chemical substance with substitutional stripping voltammetry and a sensor chip used therefor

ABSTRACT

The purpose of the invention is to provide a method for quantifying a chemical substance with high accuracy using substitutional stripping voltammetry and a sensor chip used therefor. 
     A sensor chip comprising a stripping electrode which is covered with stripping gel and a method utilizing the sensor chip. A reaction represented by the following formula (III) occurs at the stripping electrode. 
       [Chem. 3] 
       Ag+X ⊖ →AgX↓+ e   ⊖   (III)

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method for quantifying a chemical substancewith substitutional stripping voltammetry and a sensor chip usedtherefor.

2. Description of the Background Art

Patent Document 1 discloses substitutional stripping voltammetry. Thesubstitutional stripping voltammetry allows a chemical substancecontained in a solution to be electrochemically quantified with highsensitivity.

FIG. 1 shows the system for the substitutional stripping voltammetrydisclosed in the Patent Document 1.

The system comprises a pair of comb-shaped working electrodes 1, astripping electrode 2, a reference electrode 3, a counter electrode 4, asolution 5, a stripping liquid 6, a salt bridge 7, an ion conductor 8, apotentiostat 9, a recorder 10, and a switch box 11.

The solution 5 contains a chemical substance to be quantified and anoxidation-reduction substance. The stripping liquid 6 contains astandard electrolyte and a support electrolyte.

FIG. 2 shows a sensor chip 101 a employed for the substitutionalstripping voltammetry disclosed in the Patent Document 1.

The sensor chip 101 a comprises a plurality of electrodes 2 to 4 on thesurface thereof. Furthermore, the container 64 covers the surface of thesensor chip 101 a. The container 64 comprises a first penetrated opening64 a and a second penetrated opening 64 b. The solution 5 and thestripping liquid 6 are supplied to the first penetrated opening 64 a andthe second penetrated opening 64 b, respectively.

CITATION LIST Patent Literature

-   [PTL 1] Japanese Patent Publication No. 3289059B.

SUMMARY OF THE INVENTION Technical Problem

The evaporation of the stripping liquid 6 changes the concentration ofthe standard electrolyte. This causes the quantification accuracy of thechemical substance to be lowered.

The purpose of the invention is to provide a method for quantifying achemical substance with high accuracy using substitutional strippingvoltammetry and a sensor chip used therefor.

Solution to Problem

The following items [1] to [11] solve the above problem(s).

[1]: A method for quantifying a chemical substance contained in a samplesolution, comprising the following steps (a) to (e):

a step (a) of preparing a sensor chip (300), wherein,

-   -   the sensor chip comprises a substrate (30), a pair of working        electrodes (31 a/31 b), a counter electrode (33), a stripping        electrode (34), and a stripping gel (35),    -   the pair of working electrodes (31 a/31 b) is composed of a        first working electrode (31 a) and a second working electrode        (31 b),    -   the surface of the stripping electrode (34) comprises silver,    -   the stripping gel (35) covers the stripping electrode (34),    -   the stripping gel (35) does not cover the pair of the working        electrodes (31 a/31 b) or the counter electrode (33),    -   the stripping gel (35) contains a standard electrolyte and an        ionic liquid,    -   the stripping gel (35) contains no water,    -   the ionic liquid is hydrophobic and nonvolatile,    -   the ionic liquid is consisted of a cation and an anion,    -   the standard electrolyte is consisted of the cation and a halide        ion,

a step (b) of supplying the sample solution to the surface of the sensorchip to cover the surface with the sample solution, wherein,

-   -   the sample solution contains the chemical substance and an        oxidation-reduction substance or contains the chemical substance        modified with an oxidation-reduction substance,

a step (c) of applying a potential to the first working electrode (31 a)with potentiostat, and connecting the second working electrode (31 b) tothe stripping electrode (34) to generate reactions represented by thefollowing formulas (I) to (III) in the respective surface of the firstworking electrode (31 a), the second working electrode (31 b), and thestripping electrode (34), respectively,

the first working electrode (31 a):

(wherein, n represents an integer, and m represents a positive integer.)

the second working electrode (31 b):

(wherein, n represents an integer, and m represents a positive integer.)

the stripping electrode (34):

[Chem. 3]

Ag+X^(⊖)→AgX↓+e ^(⊖)  (III)

(wherein, X represents iodine atom, bromine atom, or chlorine atom.)

-   -   wherein, the silver halide is deposited on the surface of the        stripping electrode (34),

a step (d) of applying a potential to the stripping electrode (34) in acondition where no potential is applied to either the first workingelectrode (31 a) or the second working electrode (31 b) to measure acurrent flowing through the stripping electrode (34),

a step (e) of calculating the concentration of the oxidation-reductionsubstance (reductant) to quantify the chemical substance on the basis ofthe calculated concentration.

[2]: A method according to above item 1, wherein,

the sensor chip (300) further comprises a cover (37) with an inlet (36),

a space is formed between the cover (37) and the sensor chip (300),

in the step (b), the sample solution is supplied through an inlet (36)to the surface of the sensor chip (300).

[3]: A method according to above item 2, wherein,

the sensor chip (300) further comprises an air vent (38),

in the step (b), the air which the has filled the space is drainedthrough the air vent (38).

[4]: A method according to above item 2, wherein,

after the step (b), the space is filled with the sample solution.

[5]: A method according to above item 1, wherein,

the cation and the anion are selected from the following groups (I) and(II), respectively:

Group (I): a cation represented by the following formula IV-(1) toIV-(6).

(wherein, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are the same as ordifferent from each other, and represent hydrogen atom, a straight orbranched alkyl group which may contain heteroatom, an aralkyl group, oran aryl group, and R⁹, R¹⁰, R¹¹, and R¹² are the same as or differentfrom each other, and represent a straight or branched alkyl group whichmay contain heteroatom, an aralkyl group, or an aryl group.)

Group (II): an anion represented by the following formula V-(1) orV-(2).

(wherein, Rf¹ and Rf² are the same as or different from each other, andrepresent a perfluoroalkyl group having carbon number of 1 to 4.)[6]: A method according to above item 1, wherein,

-   -   the ionic liquid is selected from the following:

-   1,3-Dimethylimidazolium bis(trifluoromethanesulfonyl)imide,

-   1-Ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide,

-   1-Ethyl-3-methylimidazolium triflate,

-   1-Ethyl-3-methylimidazolium bis(pentafluoroethanesulfonyl)imide,

-   1,3-Diethylimidazolium bis(trifluoromethanesulfonyl)imide,

-   1,3-Diethylimidazolium triflate,

-   1-Butyl-3-ethylimidazolium triflate,

-   1,2-Dimethyl-3-ethylimidazolium bis(trifluoromethanesulfonyl)imide,

-   1-Butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide,

-   1-Butyl-3-methylimidazolium triflate,

-   1-isoPropyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide,

-   1,2-Dimethyl-3-propyllimidazolium    bis(trifluoromethanesulfonyl)imide,

-   N,N-propylmethylpyrrolidinium bis(trifluoromethanesulfonyl)imide,

-   Propyltrimethyammonium bis(trifluoromethanesulfonyl)imide,

-   N,N-Methylpropylpiperidinium bis(trifluoromethanesulfonyl)imide, or

-   N-Butylpyridinium bis(trifluoromethanesulfonyl)imide.    [7]: A sensor chip for a substitutional stripping voltammetry,    comprising:

a substrate (30),

a pair of working electrodes (31 a/31 b),

a counter electrode (33),

a stripping electrode (34), and

a stripping gel (35), wherein

the pair of working electrodes (31 a/31 b) is composed of a firstworking electrode (31 a) and a second working electrode (31 b),

the surface of the stripping electrode (34) comprises silver,

the stripping gel (35) covers the stripping electrode (34),

the stripping gel (35) does not cover the pair of the working electrodes(31 a/31 b) or the counter electrode (33),

the stripping gel (35) contains a standard electrolyte and an ionicliquid,

the ionic liquid is hydrophobic,

the ionic liquid is consisted of a cation and an anion,

the standard electrolyte is consisted of the cation and a halide ion.

[8]: A sensor chip according to above item 7, wherein,

the sensor chip (300) further comprises a cover (37) with an inlet (36),

a space is formed between the cover (37) and the sensor chip (300).

[9]: A sensor chip according to above item 8, wherein, the sensor chip(300) further comprises an air vent (38).[10]: A sensor chip according to above item 7, wherein,

the cation and the anion are selected from the following groups (I) and(II), respectively:

Group (I): a cation represented by the following formula IV-(1) toIV-(6).

(wherein, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are the same as ordifferent from each other, and represent hydrogen atom, a straight orbranched alkyl group which may contain heteroatom, an aralkyl group, oran aryl group, and R⁹, R¹⁰, R¹¹, and R¹² are the same as or differentfrom each other, and represent a straight or branched alkyl group whichmay contain heteroatom, an aralkyl group, or an aryl group.)

Group (II): an anion represented by the following formula V-(1) orV-(2).

(wherein, Rf¹ and Rf² are the same as or different from each other, andrepresent a perfluoroalkyl group having carbon number of 1 to 4.)[11]: A sensor chip according to above item 7, wherein,

the ionic liquid is selected from the following:

-   1,3-Dimethylimidazolium bis(trifluoromethanesulfonyl)imide,-   1-Ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide,-   1-Ethyl-3-methylimidazolium triflate,-   1-Ethyl-3-methylimidazolium bis(pentafluoroethanesulfonyl)imide,-   1,3-Diethylimidazolium bis(trifluoromethanesulfonyl)imide,-   1,3-Diethylimidazolium triflate,-   1-Butyl-3-ethylimidazolium triflate,-   1,2-Dimethyl-3-ethylimidazolium bis(trifluoromethanesulfonyl)imide,-   1-Butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide,-   1-Butyl-3-methylimidazolium triflate,-   1-isoPropyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide,-   1,2-Dimethyl-3-propyllimidazolium    bis(trifluoromethanesulfonyl)imide,-   N,N-Propylmethylpyrrolidinium bis(trifluoromethanesulfonyl)imide,-   Propyltrimethyammonium bis(trifluoromethanesulfonyl)imide,-   N,N-Methylpropylpiperidinium bis(trifluoromethanesulfonyl)imide, or-   N-Butylpyridinium bis(trifluoromethanesulfonyl)imide.

Advantageous Effects of Invention

The present invention provides a method for quantifying a chemicalsubstance with high accuracy using substitutional stripping voltammetryand a sensor chip used therefor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the system for the substitutional stripping voltammetrydisclosed in Patent Document 1.

FIG. 2 shows a sensor chip 101 a employed for the substitutionalstripping voltammetry disclosed in Patent Document 1.

FIG. 3 shows the sensor chip 300 according to the embodiment 1.

FIG. 4 schematically shows the sensor chip 300 which is connected to thepotentiostat 42.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

An embodiment of the present invention is described below with referenceto FIG. 3 and FIG. 4.

(Step (a))

First, a sensor chip 300 is prepared.

FIG. 3 shows the sensor chip 300 according to the embodiment 1. FIG. 4schematically shows the sensor chip 300 which is connected to thepotentiostat 42. The sensor chip 300 comprises a substrate 30, a pair ofcomb-shaped working electrodes 31 a/31 b, a reference electrode 32, acounter electrode 33, a stripping electrode 34, a striping gel 35, and acover 37. The cover 37 is comprised optionally, and comprises an inlet36 for injecting a sample solution and an air vent 38.

The pair of the comb-shaped working electrodes 31 a/31 b is composed ofa first working electrode 31 a and a second working electrode 31 b.

The reference electrode 32 is comprised optionally. In light of highaccuracy of the quantification, it is preferred that the sensor chip 300comprises the reference electrode 32.

The substrate 30 comprises an electrode region 30 a and a connectionregion 30 b. The cover 37 covers the electrode region 30 a on thequantification of the chemical substance, whereas the cover 37 does notcover the connection region 30 b.

The pair of comb-shaped electrodes 31 a/31 b, the reference electrode32, the counter electrode 33, and the stripping electrode 34 are formedin the electrode region 30 a. Each of these electrodes comprises a leadwire(s) 39. All the lead wires 39 are not electrically connect to eachother. In the electrode region 30 a, they are covered with an insulatorfilm (not shown), which prevents them from coming into contact with thesample solution. All the terminals of the lead wires 39 are extendedinto the connection region 30 b, which is inserted into the connector(not shown) of the potentiostat 42 shown in FIG. 4.

An example of the shape of the substrate 30 is a rectangle, a square,and an ellipse. The surface of the substrate 30 comprises an insulatorlayer (not shown). Preferably, the surface of the substrate 30 is flatin light of formation of the electrodes.

The pair of comb-shaped electrodes 31 a/31 b are disposed anywhere inthe electrode region 30 a as long as they are not connected with thestripping gel 35 and other electrodes electrically. Preferably, they aredisposed on the periphery of the center of the electrode region 30 a. Anexample of the material of the pair of comb-shaped electrodes 31 a/31 bis gold, platinum, or glassy carbon in light of stability againstelectrochemical reactions. The pair of comb-shaped electrodes 31 a/31 bface each other and are engaged.

The reference electrode 32 is also disposed anywhere in the electroderegion 30 a. Preferably, it is disposed on the periphery of the pair ofcomb-shaped electrodes 31 a/31 b. On the electrochemical measuring, thereference electrode 32 has constant potential. An example of thereference electrode 32 is a silver/silver chloride electrode.

The counter electrode 33 may be disposed anywhere in the electroderegion 30 a. The shape of the counter electrode 33 is not also limited.It is preferred that the area of the counter electrode 33 isapproximately twenty to thirty times as large as the area of the pair ofcomb-shaped electrode 31 a/31 b and the area of the stripping electrode34. An example of the material of the counter electrode 33 is gold,platinum, and glassy carbon in light of stability againstelectrochemical reactions similarly to the pair of comb-shapedelectrodes 31 a/31 b.

The stripping electrode 34 comprises silver on the surface thereof.

The stripping gel 35 covers the stripping electrode 34, which preventsthe stripping electrode 34 from coming into contact with the samplesolution. It is preferred that the stripping gel 35 is located on theperiphery of the pair of comb-shaped electrodes 31 a/31 b to lower theresistance therebetween. The stripping gel 35 is preferably a thin film.

The stripping gel 35 does not cover other electrodes 31 to 33 other thanthe stripping electrode 34. In the case that the stripping gel 35contacts with at least one of the other electrode 31 to 33, thestripping gel 35 would connect electrically to at least one electrode.This causes the quantification of the chemical substance to beimpossible.

The inlet 36 and the air vent 38 may be disposed on the top or sideplate of the cover 37. The shapes of the inlet 36 and the air vent 38are not limited. The cover 37 covers the entire of the electrode region30 a. The cover 37 prevents the sample solution supplied therein fromflowing to the connection region 30 b and out of the sensor chip 300.The cover 37 is provided optionally. The sample solution may be appliedto the surface of the sensor chip 300 without the cover 37.

Next, the stripping gel 35 is described below in more detail.

The stripping gel 35 contains the standard electrolyte and the ionicliquid. The ionic liquid serves as a support electrolyte. The strippinggel 35 is configured so as the standard electrolyte and the ionic liquidare not mixed with the sample solution. A method for configuring thestripping gel 35 is not limited. An example is supporting and/orincluding the standard electrolyte and the ionic liquid in a hydrophobicpolymer. An example of the hydrophobic polymer is poly(vinylidenefluoride-hexafluoropropylene), polymethyl methacrylate,polyacrylonitrile, and polybutylacrylate.

The ionic liquid is hydrophobic. The hydrophobic ionic liquid iscomposed of the following cation and anion.

Cation: a cation represented by the following formula IV-(1) to IV-(6).

(wherein, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are the same as ordifferent from each other, and represent hydrogen atom, a straight orbranched alkyl group which may contain heteroatom, an aralkyl group, oran aryl group, and R⁹, R¹⁰, R¹¹, and R¹² are the same as or differentfrom each other, and represent a straight or branched alkyl group whichmay contain heteroatom, an aralkyl group, or an aryl group.)

Preferably, in the imidazolium ion represented by the formula IV-(1), R¹is selected from the group consisting of methyl group, ethyl group,n-propyl group, isopropyl group, n-butyl group, isobutyl group, andt-butyl group, R² is hydrogen atom or methyl group, R³ is an alkyl grouphaving carbon number of 1 to 6 which may contain hetero atom, and R⁴ andR⁵ are hydrogen atom.

Preferably, in the isoquinolium ion represented by the formula IV-(2),R² is an alkyl group having carbon number of 1 to 6 which may containhetero atom, and R¹, R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are hydrogen atom.

Preferably, in the pyridinium ion represented by the formula IV-(3), R¹is an alkyl group having carbon number of 1 to 6 which may containhetero atom, and R², R³, R⁴, R⁵, and R⁶ are hydrogen atom.

Preferably, in the pyrrolidinium ion represented by the formula IV-(4),R¹ is selected from the group consisting of methyl group, ethyl group,n-propyl group, isopropyl group, n-butyl group, isobutyl group, andt-butyl group, R² is an alkyl group having carbon number of 1 to 6 whichmay contain hetero atom, R³, R⁴, R⁵, and R⁶ are hydrogen atom.

Preferably, in the piperidinium ion represented by the formula IV-(5),R¹ is selected from the group consisting of methyl group, ethyl group,n-propyl group, isopropyl group, n-butyl group, isobutyl group, andt-butyl group, R² is an alkyl group having carbon number of 1 to 6 whichmay contain hetero atom, and R³, R⁴, R⁵, R⁶, and R⁷ are hydrogen atom.

Preferably, in the ammonium ion represented by the formula IV-(6), R⁹,R¹⁰, R¹¹, and R¹² are the same as or different from each other, andrepresent an alkyl group having carbon number of 1 to 6 which maycontain halogen atom, a phenyl group, or a benzyl group.

Anion: an anion represented by the following formula V-(1) or V-(2).

(wherein, Rf¹ and Rf² are the same as or different from each other, andrepresents a perfluoroalkyl group having carbon number of 1 to 4.)

Preferably, in the anion represented by the formula V-(1), Rf¹ and Rf²are identical perfluoromethyl group or perfluoroethyl group.

Preferably, in the anion represented by the formula V-(2), Rf¹ istrifluoromethyl group.

More specifically, the ionic liquid is exemplified below.

-   1,3-Dimethylimidazolium bis(trifluoromethanesulfonyl)imide-   1-Ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide-   1-Ethyl-3-methylimidazolium triflate-   1-Ethyl-3-methylimidazolium bis(pentafluoroethanesulfonyl)imide-   1,3-Diethylimidazolium bis(trifluoromethanesulfonyl)imide-   1,3-Diethylimidazolium triflate-   1-Butyl-3-ethylimidazolium triflate-   1,2-Dimethyl-3-ethylimidazolium bis(trifluoromethanesulfonyl)imide-   1-Butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide-   1-Butyl-3-methylimidazolium triflate-   1-isoPropyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide-   1,2-Dimethyl-3-propyllimidazolium bis(trifluoromethanesulfonyl)imide-   N,N-Propylmethylpyrrolidinium bis(trifluoromethanesulfonyl)imide-   Propyltrimethyammonium bis(trifluoromethanesulfonyl)imide-   N,N-Methylpropylpiperidinium bis(trifluoromethanesulfonyl)imide-   N-Butylpyridinium bis(trifluoromethanesulfonyl)imide

The standard electrolyte is consisted of the above-mentioned cation anda halide ion. The halide ion denotes chloride ion, bromide ion, oriodide ion.

It is preferred that the standard electrolyte comprises identical orsimilar cation to that of the ionic liquid in light of solubility. The“similar cation” means any cation represented by the formula IV-(1),when the cation of the ionic liquid is a cation represented by theformula IV-(1). Specifically, when the ionic liquid is1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide, thestandard electrolyte may preferably be 1-butyl-3-methylimidazoliumhalide.

(Preparation Method of the Sensor Chip)

A procedure to prepare the sensor chip of the present embodiment isdescribed below.

(Formation of Electrodes)

An insulated substrate coated by a photoresist is exposed to anultraviolet through a mask for patterning, followed by alkalinedeveloping. A metal is sputtered on the patterned substrate 30. Theresidue photoresist is dissolved into an organic solvent to remove theunnecessary metal. The whole surface of the substrate 30 is coated by aninsulating film. The insulating film over the electrodes is removed bydry etching to form the pair of comb-shaped electrodes 31 a/31 b, thereference electrode 32, the counter electrode 33 and the strippingelectrode 34. The Ag/AgCl electrode is formed by applying an Ag/AgClpaste on the reference electrode 32.

(Formation of Stripping Gel 35)

The stripping gel 35 may be formed as below.

First, poly(vinylidene fluoride-hexafluoropropylene) is dissolved inacetone by ultrasonic wave on ice cooling to prepare an acetonesolution. 1-Butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imidecontaining 1-butyl-3-methylimidazolium iodide is added to the acetonesolution. Subsequently, the acetone solution is stirred and dropped onthe stripping electrode 34. Finally, the acetone is evaporated to formthe stripping gel 35.

(Step (b))

A sample solution is supplied to the surface of the above-mentionedsensor chip 300 to cover the surface with the sample solution.

The sample solution contains a chemical substance to be quantifiedaccording to the present invention. An example of the chemical substanceis an antigen, an antibody, a nucleic acid, a cell, bacteria, virus, ahapten, and a sugar.

Preferably, the cover 37 is provided with the sensor chip 300. Thesample solution supplied through the inlet 36 covers the surface of theelectrode region 30 a of the sensor chip 300. The air in the spacebetween the cover 37 and the sensor chip 300 is drained through the airvent 38.

More preferably, the space between the cover 37 and the sensor chip 300is filled with the sample solution. This allows the volume of the liquidsolution to be constant.

The sample solution contains a chemical substance to be quantified andan oxidation-reduction substance. In the present invention, the samplesolution contains the oxidation-reduction substance in the reductioncondition. The chemical substance to be quantified and anoxidation-reduction substance may be distinct. For example, the chemicalsubstance to be quantified is an enzyme, and the oxidation-reductionsubstance is an electric mediator such as potassium ferrocyanide. Or,the chemical substance to be quantified may be modified with theoxidation-reduction substance. The protein modified with aferrocenecarboxylic acid (hereinafter, “FcCOOH”) is exemplified.

The substitutional stripping voltammetry comprises a step (c) and a step(d).

(Step (c))

In the step (c), the switch 41 a and the switch 41 b are connected tothe respective terminals A, and a constant potential is applied to thefirst working electrode 31 a. Furthermore, the second working electrode31 b is electrically connected to the stripping electrode 34 to form aredox cycle between the pair of comb-shaped electrodes 31 a/31 b.

When the oxidation-reduction substance is ferrocenecarboxylic acid, thefollowing reactions represented by the following chemical formulas (VI)to (VIII) are caused on the comb-shaped electrodes 31 a, the comb-shapedelectrodes 31 b, and the stripping electrode 34.

Silver halide is deposited on the surface of the stripping electrode 34,which is composed of silver.

Comb-Shaped Working Electrode 31 a:

Comb-Shaped Working Electrode 31 b:

Stripping Electrode 34:

[Chem. 8]

Ag+X^(⊖)→AgX↓+e ^(⊖)  (VIII)

(wherein, X represents iodine atom, bromine atom, or chlorine atom.)

(Step (d))

In the step (d), the switch 41 a and the switch 41 b are connected tothe respective terminals B. No potential is applied to either the firstworking electrode 31 a or the second working electrode 31 b. Thestripping electrode 34 is swept with the potentiostat 42. This causeelectrolysis of the silver halide which has been deposited at the step(c), thus resultant halide ion is dissolved in the stripping gel asshown in the following chemical formula (IX).

Stripping Electrode 34:

[Chem. 9]

AgX+e ^(⊖)→Ag+X^(⊖)  (VIII)

(wherein, X represents iodine atom, bromine atom, or chlorine atom.)

If the stripping gel 35 contains water, the water inhibits the reactionrepresented by the formula (VIII). Because water has an affinity withthe halide ion. Therefore, the stripping gel 35 contains no water.However, the stripping gel may contain water as long as the content ofthe water is within a range of a determination precision is notadversely affected. The ionic liquid is nonvolatile. Accordingly, unlikeprior arts, the eveporation of the stripping gel 35 is suppressed in thestep (c) and in the step (d). This allows the concentration of thestandard electrolyte to be maintained. As a result, the quantificationof the chemical substance is allowed to be more accurate. Thischaracterizes the present invention.

In the longer period the constant potential is applied in the step (c),the higher sensitivity is achieved, since the deposition amount ofsilver halide is increased.

The amount of the current flowing on the dissolution in the step (d) isproportional to the deposited amount of the silver halide. The depositedamount of the silver halide is proportional to the product of theconcentration of the oxidation-reduction substance (reductant) by theperiod when the potential is applied in the step (c). Namely, thefollowing equation is satisfied.

$\begin{matrix}{\left( {{the}\mspace{14mu} {deposition}\mspace{14mu} {amount}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {silver}\mspace{14mu} {halide}} \right) = {\begin{pmatrix}{{the}\mspace{14mu} {concentration}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {oxidation}\text{-}{reduction}\mspace{14mu} {substance}} \\({reductant})\end{pmatrix} \times \begin{pmatrix}{{the}\mspace{14mu} {period}\mspace{14mu} {when}\mspace{14mu} {the}} \\{{poyenial}\mspace{14mu} {is}\mspace{14mu} {applied}\mspace{14mu} {in}\mspace{14mu} {the}\mspace{14mu} {step}\mspace{14mu} (c)}\end{pmatrix}}} & \left\lbrack {{Math}.\mspace{14mu} 1} \right\rbrack\end{matrix}$

Accordingly, the concentration of the oxidation-reduction substance(reductant) is calculated from the amount of the current flowing in thestep (d). The chemical substance is quantified on the basis of theconcentration of the oxidation-reduction substance (reductant). Needlessto say, similarly to a typical procedure, when the chemical substance isquantified from the current, a standard curve which has been prepared isused.

INDUSTRIAL APPLICABILITY

The present invention provides a method for quantifying a chemicalsubstance with high accuracy using substitutional stripping voltammetryand a sensor chip used therefor.

REFERENCE SIGNS LIST

-   -   300 sensor chip    -   30 substrate    -   30 a electrode region    -   30 b connection region    -   31 a first working electrode    -   31 b second electrode    -   32 reference working electrode    -   33 counter electrode    -   34 stripping electrode    -   35 stripping gel    -   36 inlet    -   37 cover    -   38 air vent    -   39 lead wire    -   41 switch box    -   41 a switch    -   41 b switch    -   42 potentiostat    -   W working electrode of potentiostat    -   R reference electrode of potentiostat    -   C counter electrode of potentiostat

1. A method for quantifying a chemical substance contained in a samplesolution, comprising the following steps (a) to (e): a step (a) ofpreparing a sensor chip, wherein, the sensor chip comprises a substrate,a pair of working electrodes, a counter electrode, a strippingelectrode, and a stripping gel, the pair of working electrodes iscomposed of a first working electrode and a second working electrode,the surface of the stripping electrode comprises silver, the strippinggel covers the stripping electrode, the stripping gel does not cover thepair of the working electrodes or the counter electrode, the strippinggel contains a standard electrolyte and an ionic liquid, the strippinggel contains no water, the ionic liquid is hydrophobic and nonvolatile,the ionic liquid is consisted of a cation and an anion, the standardelectrolyte is consisted of the cation and a halide ion, a step (b) ofsupplying the sample solution to the surface of the sensor chip to coverthe surface with the sample solution, wherein, the sample solutioncontains the chemical substance and an oxidation-reduction substance orcontains the chemical substance modified with an oxidation-reductionsubstance, a step (c) of applying a potential to the first workingelectrode with potentiostat, and connecting the second working electrodeto the stripping electrode to generate reactions represented by thefollowing formulas (I) to (III) in the respective surface of the firstworking electrode, the second working electrode, and the strippingelectrode, respectively, the first working electrode:

(wherein, n represents an integer, and m represents a positive integer.)the second working electrode:

(wherein, n represents an integer, and m represents a positive integer.)the stripping electrode:[Chem. 3]Ag+X^(⊖)→AgX↓+e ^(⊖)  (III) (wherein, X represents iodine atom, bromineatom, or chlorine atom.) wherein, the silver halide is deposited on thesurface of the stripping electrode, a step (d) of applying a potentialto the stripping electrode in a condition where no potential is appliedto either the first working electrode or the second working electrode tomeasure a current flowing through the stripping electrode, a step (e) ofcalculating the concentration of the oxidation-reduction substance(reductant) to quantify the chemical substance on the basis of thecalculated concentration.
 2. A method according to claim 1, wherein, thesensor chip further comprises a cover with an inlet, a space is formedbetween the cover and the sensor chip, in the step (b), the samplesolution is supplied through an inlet to the surface of the sensor chip(300).
 3. A method according to claim 2, wherein, the sensor chipfurther comprises an air vent, in the step (b), the air which the hasfilled the space is drained through the air vent.
 4. A method accordingto claim 2, wherein, after the step (b), the space is filled with thesample solution.
 5. A method according to claim 1, wherein, the cationand the anion are selected from the following groups (I) and (II),respectively: Group (I): a cation represented by the following formulaIV-(1) to IV-(6).

(wherein, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are the same as ordifferent from each other, and represent hydrogen atom, a straight orbranched alkyl group which may contain heteroatom, an aralkyl group, oran aryl group, and R⁹, R¹⁰, R¹¹, and R¹² are the same as or differentfrom each other, and represent a straight or branched alkyl group whichmay contain heteroatom, an aralkyl group, or an aryl group.) Group (II):an anion represented by the following formula V-(1) or V-(2).

(wherein, Rf¹ and Rf² are the same as or different from each other, andrepresent a perfluoroalkyl group having carbon number of 1 to 4.)
 6. Amethod according to claim 1, wherein, the ionic liquid is selected fromthe following: 1,3-Dimethylimidazoliumbis(trifluoromethanesulfonyl)imide, 1-Ethyl-3-methylimidazoliumbis(trifluoromethanesulfonyl)imide, 1-Ethyl-3-methylimidazoliumtriflate, 1-Ethyl-3-methylimidazoliumbis(pentafluoroethanesulfonyl)imide, 1,3-Diethylimidazoliumbis(trifluoromethanesulfonyl)imide, 1,3-Diethylimidazolium triflate,1-Butyl-3-ethylimidazolium triflate, 1,2-Dimethyl-3-ethylimidazoliumbis(trifluoromethanesulfonyl)imide, 1-Butyl-3-methylimidazoliumbis(trifluoromethanesulfonyl)imide, 1-Butyl-3-methylimidazoliumtriflate, 1-isoPropyl-3-methylimidazoliumbis(trifluoromethanesulfonyl)imide, 1,2-Dimethyl-3-propyllimidazoliumbis(trifluoromethanesulfonyl)imide, N,N-propylmethylpyrrolidiniumbis(trifluoromethanesulfonyl)imide, Propyltrimethyammoniumbis(trifluoromethanesulfonyl)imide, N,N-Methylpropylpiperidiniumbis(trifluoromethanesulfonyl)imide, or N-Butylpyridiniumbis(trifluoromethanesulfonyl)imide.
 7. A sensor chip for asubstitutional stripping voltammetry, comprising: a substrate, a pair ofworking electrodes, a counter electrode, a stripping electrode, and astripping gel, wherein the pair of working electrodes is composed of afirst working electrode and a second working electrode, the surface ofthe stripping electrode comprises silver, the stripping gel covers thestripping electrode, the stripping gel does not cover the pair of theworking electrodes or the counter electrode, the stripping gel containsa standard electrolyte and an ionic liquid, the ionic liquid ishydrophobic, the ionic liquid is consisted of a cation and an anion, thestandard electrolyte is consisted of the cation and a halide ion.
 8. Asensor chip according to claim 7, wherein, the sensor chip furthercomprises a cover with an inlet, a space is formed between the cover andthe sensor chip.
 9. A sensor chip according to claim 8, wherein, thesensor chip further comprises an air vent.
 10. A sensor chip accordingto claim 7, wherein, the cation and the anion are selected from thefollowing groups (I) and (II), respectively: Group (I): a cationrepresented by the following formula IV-(1) to IV-(6).

(wherein, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are the same as ordifferent from each other, and represent hydrogen atom, a straight orbranched alkyl group which may contain heteroatom, an aralkyl group, oran aryl group, and R⁹, R¹⁰, R¹¹, and R¹² are the same as or differentfrom each other, and represent a straight or branched alkyl group whichmay contain heteroatom, an aralkyl group, or an aryl group.) Group (II):an anion represented by the following formula V-(1) or V-(2).

(wherein, Rf¹ and Rf² are the same as or different from each other, andrepresent a perfluoroalkyl group having carbon number of 1 to 4.)
 11. Asensor chip according to claim 7, wherein, the ionic liquid is selectedfrom the following: 1,3-Dimethylimidazoliumbis(trifluoromethanesulfonyl)imide, 1-Ethyl-3-methylimidazoliumbis(trifluoromethanesulfonyl)imide, 1-Ethyl-3-methylimidazoliumtriflate, 1-Ethyl-3-methylimidazoliumbis(pentafluoroethanesulfonyl)imide, 1,3-Diethylimidazoliumbis(trifluoromethanesulfonyl)imide, 1,3-Diethylimidazolium triflate,1-Butyl-3-ethylimidazolium triflate, 1,2-Dimethyl-3-ethylimidazoliumbis(trifluoromethanesulfonyl)imide, 1-Butyl-3-methylimidazoliumbis(trifluoromethanesulfonyl)imide, 1-Butyl-3-methylimidazoliumtriflate, 1-isoPropyl-3-methylimidazoliumbis(trifluoromethanesulfonyl)imide, 1,2-Dimethyl-3-propyllimidazoliumbis(trifluoromethanesulfonyl)imide, N,N-Propylmethylpyrrolidiniumbis(trifluoromethanesulfonyl)imide, Propyltrimethyammoniumbis(trifluoromethanesulfonyl)imide, N,N-Methylpropylpiperidiniumbis(trifluoromethanesulfonyl)imide, or N-Butylpyridiniumbis(trifluoromethanesulfonyl)imide.